Synthesis, Antibacterial and Anthelmintic Activity of Novel 3-(3-Pyridyl)-oxazolidinone-5-methyl Ester Derivatives

In this study, a series of 3-(3-pyridyl)-oxazolidone-5-methyl ester derivatives was synthesized and characterized by 1H NMR, 13C NMR, and LC-MS. The conducted screening antibacterial studies of the new 3-(3-pyridyl)-oxazolidone-5-methyl ester derivatives established that the methyl sulfonic acid esters have broad activity spectrum towards Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus epidermidis. Among them, compound 12e has the most potent activity, with an MIC of 16 μg/mL against B.subtilis, and could reduce the instantaneous growth rate of bacteria. Furthermore, molecular docking studies were also simulated for compound 12e to predict the specific binding mode of this compound. In addition, anthelmintic activity of these compounds was also evaluated against adult Indian earthworms (Pheretima posthuman). The results showed that compound 11b had the best effect. These results above can provide experimental reference for the development of novel antibacterial and anthelmintic drugs.


Introduction
With bacterial resistance comes a grave threat to global public health security. Owing to the rapid growth of clinical drug-resistant bacteria, the number of effective anti-infective drugs has declined, and patient mortality is increasing [1][2][3][4][5]. It is estimated that about 10 million people all over the world will die from drug-resistant bacterial infections, which will become the leading cause of human disease death, and lead to the loss of global GDP of US $100 trillion by 2050 [6]. Therefore, how to effectively deal with the bacterial resistance crisis is a challenging question. Many new therapies, such as those utilizing nanoparticles, phages, and protic ionic liquids, are being developed [7][8][9][10][11]. However, these technologies are still immature and have not been effectively used clinically [12][13][14]. At present, chemosynthesized antibacterial agents still play an indispensable role in clinical practice. Therefore, for pharmaceutical chemists all over the world, the discovery of novel active antibacterial compounds is an urgent mission and the direction of continuous efforts.
Linezolid, an oxazolidinone antibacterial agent, has been widely used in the treatment of infection caused by multi-drug-resistant Gram-positive bacteria since it was approved for clinical use in 2000. However, with clinical use gradually increasing, many linezolidresistant strains have also been reported [15][16][17][18]. To find novel, safe and effective antibacterial drugs, there have been many studies in recent years on the structural modification of linezolid [19][20][21][22][23][24][25]. Among them, many have focused on the modification of five-position side chains of oxazolidine and the morpholine ring, while a few have explored the benzene ring of linezolid. In addition, anthelmintic properties of linezolid derivatives have also been found [26].
The pyridine heterocyclic ring has been widely used in the medicine field because of its distinctive aromatic and electronegative properties [27][28][29][30][31]. In our previous studies, a series of pyridine heterocyclic derivatives were evaluated to have good biological activities [32][33][34]. Among them, compound 1 showed good antibacterial and anthelmintic activities in vitro. Based on previous research and theory [35], here we replaced the benzene ring with pyridine and a nitrogen atom with an oxygen atom at the 5-position side chain of the oxazolidone ring. A series of 3-(3-pyridyl)-oxazolidinone-5-methyl ester derivatives were designed and synthesized (Figure 1), and their antibacterial and anthelmintic effects in vitro were evaluated.

Chemistry
The general synthetic route for the target compounds is illustrated in Scheme 1. The commercially available 2-chloro-5-nitropyridine was reacted with morpholine to produce intermediate 2. Then, the nitro group was reduced to prepare amine 4. After the introduction of the Cbz group, intermediate 6 was cyclized with (R)-(-)-butylglycidyl ester under the condition of −78 • C and n-butyllithium (n-BuLi) to produce oxazolidinone intermediate 9. Finally, ester derivatives 11a-i were prepared with intermediate 9 and different acids, sulfonyl chlorides and isocyanates. Compounds 12a-h were obtained by similar methods. Scheme 1. Synthesis route of the target compounds.

Screening of Antibacterial Activity
All twelve synthesized compounds were tested against four Gram-positive strains, including S. aureus (ATCC25923), S. pneumoniae (ATCC49619), B. subtilis (BNCC109047) and S. epidermidis (BNCC186652) by broth dilution method with linezolid as the standard drug, and the MIC values are listed in Table 1. Initially, keeping the morpholine ring of linezolid in our structure, compounds 11a-i were synthesized and evaluated for antibacterial activity against four gram-positive strains. Unfortunately, the activity of most compounds was generally weak, with only compounds 11f and 11h showing a broad-spectrum effect.
Therefore, while maintaining the methyl sulfonyl group on the C-5 side chain, compounds 12a-h were synthesized by replacing the morpholine ring with a piperazine ring and modifying the side chain further. Their activity was significantly improved and they all had broad-spectrum antibacterial action. Among these compounds, compounds 12a and 12e had the best antibacterial activity, with the lowest MIC values reaching 16 µg/mL, indicating that the flexible substituents linked with piperazine had stronger antibacterial activity. The activity of 12c and 12d was slightly weaker than that of 12a and 12e, probably due to the presence of long-chain substituents, which were less binding than flexible rings, but their long chains were morphologically consistent with the target. Conversely, rigid rings, such as 12b, reduced antibacterial activity. Compared with our previous work, these compounds still retain antibacterial activity after changing the core structure to oxazolidinone [34]. Here the antibacterial activity of the synthesized compounds was not as good as linezolid, which may be due to its reduced affinity with the target caused by the structural changes. In addition, a bacterial inhibitory zone test of 12e against S. aureus was provided to visually observe bacteriostatic effects of 12e ( Figure 2). It can be seen from the figure that the bacteriostatic zone increased with the increase of drug concentration, and the diameter of the zone was similar to that of linezolid at a concentration of 20 × MIC.

Bacterial Growth Kinetic Study
The growth curve of bacteria directly shows the process and rule of the change of bacterial concentration with time, while the growth inhibition curve of bacteria can directly reflect the influence of drugs on the growth process of bacteria [36]. The growth inhibition curve of compound 12e against Bacillus subtilis is shown in Figure 3. Without treatment (red) or the presence of 1/4 MIC (blue) of 12e, the bacteria increased sharply in the log phase (2-10 h). With the increase in concentration, the lag phase was prolonged and the duration of the logarithmic phase was shortened. The significantly change was that the decreased instantaneous growth rate led to the plateau phase concentration reduction, which was manifested by the difference of the final turbidity of bacterial liquid. Until 2 × MIC of 12e (green) and linezolid (yellow), there was no obvious growth of bacteria.
These findings revealed that 12e was an excellent bactericidal agent in a concentrationdependent manner, and mainly disturbed the instantaneous growth rate of bacteria in the logarithmic phase.

Binding Mode Study
To explore the mechanism of the active compound 12e, Auto-Dock software was used to perform docking analysis with the 50S ribosomal subunit (PDB ID: 3CPW).
The docking result from Figure 4 demonstrated that 12e was inclined to locate in a long and narrow channel with a fully extended state. The five-position side chain of the oxazolidine ring was binding to a relatively small cavity (yellow), which could only accommodate a small group. This might be the reason for the moderate activity of compound 11h. In addition, it was discovered that the 4 -position side chain of the piperazine ring extended into a deep cavity (red), which could accommodate bigger structural groups after containing the flexible cyclohexyl group. Presumably, compounds 12a-h had better antibacterial effect than 11a-i, because they were suitable for the narrow cavity (yellow), and occupied the larger and deeper region (red) at the same time, making them more closely combined with the target. The specific mechanism of action is shown in Figure 5; the oxygen atoms of the sulfonyl group formed two hydrogen bonds with A2473. It followed that the sulfonyl group presumedly had better ability to form hydrogen bonds than the carbonyl group, which explained why 11h had the best antibacterial activity among 11a-i. In addition, the piperazine-linked urea group of 12e extended into the deep cavity forming a hydrogen bond with G2283, making 12b more effective.

Anthelmintic Activity Assay
Anthelmintic activity assay of 17 target compounds was performed using adult Indian earthworm (Pheretima posthuman) with albendazole as the positive standard drug. Efficacy was assessed by counting the paralysis and death time, and the results are shown in Table 2.
For compounds 11a-i, the paralysis time of most compounds was less than 15 min, and the death time was more than 50 min, indicating that these compounds had significant paralysis effect on earthworms, but poor insecticidal action. In terms of shorter paralysis time (3.6 ± 0.2 min) and death time (8.1 ± 0.8 min), the anthelmintic effect of compound 11b was better than albendazole and our previous compounds, indicating that it has potential to be developed into a new anthelmintic drug [34]. For compounds 12a-h, the paralysis time was more than 15 min and death time was not discovered within 50 min, indicating that these compounds had poor anthelmintic activity, even though they had good antibacterial activity.

Chemical Reagents and Instruments
Before using, tetrahydrofuran (THF) was dried with sodium under reflux, dichloromethane (DCM) was dried with CaH 2 under reflux and triethylamine (TEA) was dried with molecular sieve. All the reagents above were chemically pure (Tianjin Tianli Chemical Reagent Co., Ltd., Tianjin, China). All the other reagents and compounds used in this study were commercially available analytically pure (AR) without further purification.
All the chemical reactions were detected by thin-layer chromatography (TLC), which was conducted on silica gel G plates (Taizhou Luqiao Sijia Biochemical Plastic Products Factory, Taizhou, China). The plates were observed under a Zf-2 tri-use ultraviolet analyzer (Shanghai Anting Electronic Instruments Factory, Shanghai, China). Most of the reaction mixtures were purified by column chromatography carried out on 200-300 mesh sieve specifications Silica Gel (Qingdao Ocean Chemical Co., Ltd., Qingdao, China).
All the purified compounds underwent the following tests to verify their purity and structure: a WRS-1A melting point apparatus (Shanghai Shinuo Physical Optical Instrument Co., Ltd., Shanghai, China) was used to assess the compounds' melting points. The 1 H-NMR and 13 C-NMR of each compound were recorded on an NMR spectrometer (Varian, Salt Lake City, UT, USA) in DMSO-d 6 with tetramethyl-silane (TMS) as an internal standard. The mass spectra (MS) of each compound were registered in an Agilent 1100 liquid chromatography-mass spectrometry (LC/MS) instrument (Agilent Technologies Co., Ltd., Santa Clara, CA, USA).
In addition, all the work in the biological activity test was completed in a DL-CJ-2N highperformance aseptic test stand (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., Beijing, China). All biological media such as Mueller-Hinton (MH) broth medium, Luria-Bertani (LB) broth medium and Nutrient Agar (Qingdao High-tech Park Haibo Biotech-nology Co., Ltd., Qingdao, China) were all dissolved directly with deionized water then sterilized by a vertical autoclave (Zealway Instrument Co., Ltd., Xiamen, China). Bacteria used in this experiment were grown in an MCO-20AIC carbon dioxide incubator (SANYO Co., Ltd., Osaka, Japan).

Synthesis of the Target Compounds
Intermediates 2-10 were synthesized according to the steps in the literature [31][32][33][34]  Raw data for the above products are presented in Supplementary Materials (Figures S1-S16).

Minimum Inhibitory Concentration (MIC) Study
According to the standard procedure recommended by the Clinical Laboratory Standards Institute (CLSI) [37], the minimum inhibitory concentration (MIC) in vitro of all synthesized compounds for Gram-positive strains was evaluated on 96-well microplates by the broth microdilution technique to screen for antibacterial activities. In Mueller-Hinton (MH) sterile medium, a series of compound solutions was prepared by double-fold dilution method from 0.5 to 256 µg/mL. After that, 100 µL diluted standard inoculum (1 × 10 6 CFU/mL) was added into the gradient solution. The 96-well plates were cultured at 37 • C for 16-18 h, and the MIC of each well was considered to be the lowest concentration that could significantly inhibit the growth of bacteria. The untreated inoculum was used as a negative control and linezolid was used as a positive control [38].

Bacterial Inhibitory Zone Test
The bacterial inhibitory zone of 12e against S. aureus (ATCC 25923) was determined by pore diffusion method [39]. A total of 100 µL of fresh culture was dispersed on sterile petri dishes containing nutrient AGAR media. The holes were made in the petri plates using 100 µL micro pipette tips and injected with varied concentrations of 12e (160, 320, 640, and 1600 µg/mL) and 40 µg/mL of control (linezolid), following which the petri plates were kept overnight at 37 • C. After incubation, a band was formed around the holes by inhibiting biological growth.

Growth Kinetic Study
Based on the previous result of antibacterial activity, compound 12e was selected for further research. According to the literature [36], the growth kinetics of Bacillus subtilis (BNCC 109047) was determined after treatment with compound 12e and linezolid. The log-phase strain was diluted to 10 6 -10 7 CFU/mL in LB broth containing compound 12e at a series of concentrations (0, 1/4, 1/2, 1 and 2 × MIC), then incubated at 37 • C and 150 rpm. Linezolid at the concentration of 2 µg/mL was used as a positive control. At the beginning, culture mixture (3 mL) was removed from each sample solution at intervals of 1 h, and turbidity was measured by ultraviolet spectrophotometer at 600 nm. When the O.D. value became stable, the time interval was gradually increased and the time-kill curve was drawn.

Binding Mode Study
Molecular docking assay was performed using the AutoDock 4.2.6 ® software. The X-ray crystal 3D structure of the 50S ribosomal subunit (PDB ID: 3CPW) was obtained from the Protein Data Bank (PDB) database. The ligands and proteins were removed from the structure and RNA chains were retained using PyMOL 1.5.0.3. The modified structure was prepared for further molecular docking. 2D structures of the ligands were built with the aid of ChemDraw 19.0 and the 3D structures were established by Chem3D 19.0 software. The docking results were analyzed and visualized with PyMOL 1.5.0.3.

Anthelmintic Activity Assay
Due to the high similarity between adult Indian earthworms (P. posthuma) and intestinal roundworms (A. bricoides) in terms of anatomical and physiological characteristics [40], anthelmintic activities of all compounds were evaluated [41,42].
Before use, the earthworms with an average length of 6-10 cm and width of 0.2-0.3 cm were cleaned with normal saline solution [41], and the target compounds and positive control drug (albendazole) were dissolved with the minimum amount of DMSO and diluted with normal saline solution.
Then, the tested solution with a massive concentration of 2 g/L was prepared and placed in a Petri dish (d = 6 cm). After being divided into different groups with six (6) worms, they were transferred to Petri dishes with sterile forceps and the paralysis and death time were monitored and recorded with stopwatches for 1 h. Earthworms tested in standard saline (0.9% w/v) were taken as the control group. The paralysis time was considered as the time when the worm showed no obvious movement at rest or under mild vibration. In a similar way, death time was considered when there was no obvious movement after severe shaking and exposure to 50 • C water [41,42]. The mean paralysis and death times were was used to reflect the anthelmintic effect of these compounds; the shorter time, the better the effect.

Conclusions
Seventeen novel 3-(3-pyridyl)-oxazolidone-5-methyl derivatives were designed and synthesized, and their structures were identified by 1 H NMR, 13 C NMR and MS. The primary screening of antibacterial activity, which was tested on Staphylococcus aureus (ATCC25923), Streptococcus pneumoniae (ATCC49619), Bacillus subtilis (BNCC109047) and Staphylococcus epidermidis (BNCC186652), showed that most of the compounds had certain antibacterial activity, among which compound 12a and 12e had the best antimicrobial activity. In a further bactericidal kinetic assay, 12e was mainly interfering in the logarithmic phase of bacteria in a concentration-dependent manner. Molecular docking between the 12e and the 50S ribosomal subunit was studied to predict the mechanism of action. In addition, the anthelmintic activity test demonstrated that the compound 11b had the most significant anthelmintic effect. These results provide a preliminary basis for the development of novel pyridine heterocyclic compounds as antibacterial and anthelmintic agents. Further structural optimization and biological activity confirmation of the action model are in progress. Figure S1: Structure and Numbering of 11a, Figure S2: 1 H NMR Spectrum of 11a, Figure S3: 13 C NMR Spectrum of 11a. Figure S4: ES-MS for compound 11a, Figure S5: 1 H NMR Spectrum of 11b, Figure S6: 13 C NMR Spectrum of 11b. Figure S7: ES-MS for compound 11b, Figure S8: 1 H NMR Spectrum of 11c, Figure S9: 13 C NMR Spectrum of 11c. Figure S10: ES-MS for compound 11c, Figure S11: 1 H NMR Spectrum of 11d, Figure S12: 13 C NMR Spectrum of 11d. Figure S13: ES-MS for compound 11d, Figure S14: 1 H NMR Spectrum of 11e, Figure S15: 13 C NMR Spectrum of 11e. Figure S16: ES-MS for compound 11e, Figure S17: 1 H NMR Spectrum of 11f, Figure S18: 13 C NMR Spectrum of 11f. Figure S19: ES-MS for compound 11f, Figure S20: 1 H NMR Spectrum of 11g, Figure S21: 13 C NMR Spectrum of 11g. Figure S22: ES-MS for compound 11g, Figure S23: 1 H NMR Spectrum of 11h, Figure S24: 13 C NMR Spectrum of 11h. Figure S25: ES-MS for compound 11h, Figure S26: 1 H NMR Spectrum of 11i, Figure S27: 13 C NMR Spectrum of 11i. Figure S28: ES-MS for compound 11i, Figure S29: 1 H NMR Spectrum of 12a, Figure S30: 13 C NMR Spectrum of 12a. Figure S31: ES-MS for compound 12a, Figure S32: 1 H NMR Spectrum of 12b, Figure S33: 13 C NMR Spectrum of 12b. Figure S34: ES-MS for compound 12b, Figure S35: 1 H NMR Spectrum of 12c, Figure S36: 13 C NMR Spectrum of 12c. Figure S37: ES-MS for compound 12c, Figure S38: 1 H NMR Spectrum of 12d, Figure S39: 13 C NMR Spectrum of 12d. Figure S40: ES-MS for compound 12d, Figure S41: 1 H NMR Spectrum of 12e, Figure S42: 13 C NMR Spectrum of 12e. Figure S43: ES-MS for compound 12e, Figure S44: 1 H NMR Spectrum of 12f, Figure S45: 13 C NMR Spectrum of 12f. Figure S46: ES-MS for compound 12f, Figure S47: 1 H NMR Spectrum of 12g, Figure S48: 13 C NMR Spectrum of 12g. Figure S49: ES-MS for compound 12g, Figure S50: 1 H NMR Spectrum of 12h, Figure S51: 13